Intracellular Observation of RNA Metabolism
Will Help Identify Disease-Associated RNAs

(Philadelphia, PA) - For the first time, researchers
can now peer inside intact cells to not only identify RNA-binding
proteins, but also observe--in real-time--the intricate activities
of these special molecules that make them key players in managing
some of the cell's most basic functions. Researchers at the University
of Pennsylvania School of Medicine who developed the new
technology see this advance as one of the next logical steps in
genomics research. Senior author James Eberwine, PhD,
Professor of Pharmacology at Penn, and colleagues published their
research last week in the Proceedings of the National Academy
of Sciences.

“Now we have a workable system to understand all aspects of
RNA metabolism in a cell,” say Eberwine. “For the first
time, we can study how manipulation of cellular physiology, such
as administering a drug, changes RNA-binding protein and RNA interactions.
This technology allows us to see that in real time in real cells.”

RNA is the genetic material that programs cells to make proteins
from DNA’s blueprint and specifies which proteins should be
made. There are many types of RNA in the cells of mammals, such
as transfer RNA, ribosomal RNA, and messenger RNA-each with a specific
purpose in making and manipulating proteins.

The workhorses of the cell, RNA-binding proteins regulate every
aspect of RNA function. Indeed, RNA is transported from one site
to another inside the cell by RNA-binding proteins; RNA is translated
into protein with the help of RNA-binding proteins, and RNA-binding
proteins degrade used RNA. “They’re really the master
regulators of expression in the cell,” says Eberwine.

Using whole neurons from rodents, the researchers were able to identify
RNA interactions in live cells. In collaboration with Ûlo
Langel from Stockholm University, the Penn investigators devised
a many-talented molecule that does not get broken down by enzymes
once inside a live cell. One end of the molecule, called a peptide
nucleic acid (PNA), has a cell-penetrating peptide called transportan
10 to first get the PNA through the cell membrane. Once in the cell,
the PNA binds to a specific target messenger RNA (mRNA). There is
also a compound on the molecule that can be activated by light and
will cross-link the PNA to whatever protein is nearby. The researchers
isolated an array of proteins from the complex of the PNA, the targeted
mRNAs, and associated RNA-binding proteins. The cells are then broken
apart and the proteins that interact with the mRNA are identified
with a mass spectrometer.

With their system, the researchers are trying to identify RNA-binding
proteins that bind RNAs of interest-such as those involved in the
targeting, degradation, and translation of RNAs into proteins. Once
identified, the Eberwine team uses another technology they developed
to find the other RNA cargos that bind to that RNA-binding protein.
These are other RNAs that likely co-regulate RNAs associated with
disease.

The research was supported by grants from the National Institutes
of Health, the Swedish Science Foundation, and the European Community.
Study coauthors are Jennifer Zielinski, Tiina Peritz, Jeanine Jochems,
Theresa Kannanayakal, and Kevin Miyashiro, from Penn, and Kalle
Kilk, Emilia Eiriksdóttir, and Ûlo Langel from Stockholm
University, Sweden.

###

PENN Medicine is a $2.7 billion enterprise
dedicated to the related missions of medical education, biomedical
research, and high-quality patient care. PENN Medicine consists
of the University of Pennsylvania School of Medicine (founded in
1765 as the nation's first medical school) and the University of
Pennsylvania Health System.

Penn’s School of Medicine is ranked #2 in the nation
for receipt of NIH research funds; and ranked #4 in the nation in
U.S. News & World Report’s most recent ranking of top
research-oriented medical schools. Supporting 1,400 fulltime faculty
and 700 students, the School of Medicine is recognized worldwide
for its superior education and training of the next generation of
physician-scientists and leaders of academic medicine.

The University of Pennsylvania Health System comprises: its
flagship hospital, the Hospital of the University of Pennsylvania,
consistently rated one of the nation’s “Honor Roll”
hospitals by U.S. News & World Report; Pennsylvania Hospital,
the nation's first hospital; Penn Presbyterian Medical Center; a
faculty practice plan; a primary-care provider network; two multispecialty
satellite facilities; and home health care and hospice.